Process for inhibiting serine proteases using cresol or 3-hydroxypyridine and sulfonic acid derivatives or fluorophosphonates

ABSTRACT

A process is provided for inhibiting serine proteases in a sample. The process involves adding to the sample a compound such as cresol or 3-hydroxypyridine and then adding to the sample a sulfonic acid derivative or a fluorophosphonate, to derivative the serine proteases. The compounds such as cresol or 3-hydroxypyridine avoid nonspecific derivatization of proteins other than the serine proteases.

DESCRIPTION

The present invention concerns a process for inhibiting serine proteasesby derivatization with sulfonic acid derivatives or fluorophosphates, areagent for avoiding unspecific derivatization of other proteins duringthe inhibition of serine proteases by sulfonic acid derivatives orfluorophosphates as well as a reagent kit for the specific inhibition ofserine proteases.

The catalytically active binding pocket of serine proteases can bespecifically labelled and hence inhibited by sulfonic acid derivatives,and in particular by fluorides or fluorophosphates thereof. This can, onthe one hand, be used to determine the protease category (serine,cysteine, metallo, aspartyl protease) and, on the other hand, to protectproteins from proteolytic degradation (Markwardt in Pharmazie 26 (1971),401-404, J. Kraut, Ann. Rev. Biochem. (1977), vol. 46, p. 331-358).

Although the selectivity of the reaction is very high, unspecificsecondary reactions with other proteins can be occasionally detectede.g. when using the inhibitors AEBSF (4-(2-aminoethyl)-phenyl-sulfonylfluoride), PMSF (phenylmethyl-sulfonyl fluoride), diisopropylfluorophosphate (DFP) or amidinophenylmethylsulfonyl fluoride (APMSF).These depend above all on the pH conditions of the reaction, the proteinconcentration, the sulfonyl fluoride or fluorophosphate concentrationand the type of accessible amino acids. Thus at high pH values,reactions can occur at the amino terminus, on lysines and on tyrosinesof proteins. Such secondary reactions are of course especially undesiredfor proteins which are used therapeutically or diagnostically.Furthermore this can lead to falsifications of the measurement results(e.g. determination of the protein concentration by absorption at 280nm). The secondary reactions can for example be detected by sequenceanalysis or simply by mass spectroscopic analyses.

Methods Enzymol. 182 (1990), p. 87, 88 and 180, 181 describes a methodfor detecting protease contaminations in protein preparations andmentions protease inhibitors together with information on concentrationranges in which such protease inhibitors are to be used. Processes orreagents for inhibiting an unspecific derivatization of proteins whenusing protease inhibitors are not disclosed.

In a further document of the prior art (Database Biol. Abs., 89: 451839)the inhibitory effect on serine proteases and in particular on trypsinand α-chymotrypsin of 2-ethyl-6-methyl-3-oxypyridine antioxidantsphosphorylated on the oxy group is described. An increase in thespecificity of protease inhibitors is not disclosed.

Hence the object of the present invention was to provide a method foravoiding unspecific secondary reactions when serine proteases areinhibited by sulfonic acid derivatives and fluorophosphates.

This object is achieved by a process for inhibiting serine proteases byderivatization with sulfonic acid derivatives or fluorophosphates whichis characterized in that, in order to avoid unspecific derivatization ofother proteins, a compound of the general formula (I)

is added in which

X denotes C or N,

R₁ denotes H, methyl, ethyl or OH,

R₂ and R₃ each denote H, methyl, ethyl, OH or residues which togetherform a ring closure with a maximum of 6 C atoms in the ring and

R₄ denotes H, OH or an optionally substituted alkyl, alkenyl or alkinylgroup with up to 4 C atoms provided that at least one of R₁, R₂, R₃ orR₄ represents an OH group and the compound has a pKa of at least 8.

It has surprisingly turned out that the addition of compounds of formula(I) enables unspecific derivatization of proteins other than serineproteases to be practically completely prevented. The requirements whichthe compounds of formula (I) have to fulfill for this are primarily thestated pKa value since compounds with a lower pKa value do notadequately prevent the unspecific secondary reactions. Furthermore theoptional residues on the groups R₁, R₂, R₃ and R₄ and the possible ringclosure by the residues R₂ and R₃, should not allow the compound toreach a size which makes it ineffective due to steric hindrance.Attainment of the maximum size of the residues can be easily establishedby simple preliminary experiments. Within the scope of the presentinvention it is preferred to use a cresol in particular p-cresol or3-hydroxypyridine as the compound of formula (I). Both compounds areextremely effective.

The process according to the invention is therefore preferably used toprocess fermentations, culture broths and cell lysates; i.e. onlyinterfering serine proteases are irreversibly inhibited without anunspecific derivatization of the desired protein occurring. This is forexample advantageous when producing proteins which are intended fortherapeutic measures if the protein is obtained by genetic engineeringand the cell lysate is treated with sulfonic acid derivatives orfluorophosphates to avoid loss of activity by serine proteases. Thecompound of the general formula (I) added in the process according tothe invention has in addition a low molecular weight and can thus easilybe subsequently separated during the purification e.g. by dialysis.

A further subject matter of the present invention is a reagent foravoiding unspecific derivatization of proteins when serine proteases areinhibited by sulfonic acid derivatives or fluorophosphates to which acompound of the general formula (I)

is added in which

X denotes C or N,

R₁ denotes H, methyl, ethyl or OH,

R₂ and R₃ each denote H, methyl, ethyl, OH or residues which togetherform a ring closure with a maximum of 6 C atoms in the ring and

R₄ denotes H, OH or an optionally substituted alkyl, alkenyl or alkinylgroup with up to 4 C atoms provided that at least one of R₁, R₂, R₃ orR₄ represents an OH group and the compound has a pKa of at least 8.

In a particularly preferred embodiment the reagent according to theinvention contains a cresol, in particular p-cresol, or3-hydroxypyridine.

As already mentioned above, the reagent according to the invention canbe used advantageously in the processing of fermentations and culturebroths in which serine proteases are present and where it is intended toinhibit these by sulfonic acid derivatives in order to avoid losses ofdesired proteins.

A further subject matter of the present invention is a reagent kit whichcontains a) a sulfonic acid derivative or fluorophosphate and b) acompound of the general formula (I)

in which

X denotes C or N,

R₁ denotes H, methyl, ethyl or OH,

R₂ and R₃ each denote H, methyl, ethyl, OH or residues which togetherform a ring closure with a maximum of 6 C atoms in the ring and

R₄ denotes H, OH or an optionally substituted alkyl, alkenyl or alkinylgroup with up to 4 C atoms provided that at least one of R₁, R₂, R₃ orR₄ represents an OH group and the compound has a pKa of at least 8.

Such a reagent kit can be used advantageously for the inhibitionaccording to the invention of serine proteases by derivatization withsulfonic acid derivatives or fluorophosphates to avoid unspecificderivatization of other proteins. In particular interfering proteasescan be irreversibly inhibited in culture broths by this means as alreadymentioned. It is therefore advantageous to provide a reagent kitaccording to the invention as a package unit in order to carry out theprocess according to the invention. In a particularly preferredembodiment the reagent kit according to the invention contains AEBSF,APMSF or PMSF as the sulfonic acid derivative or a fluorophosphate suchas DFP and a cresol in particular or 3-hydroxy-pyridine as b). Bothcomponents of the reagent kit a) and b) must be present in separatevessels since they would otherwise already react together beforecarrying out the actual process and their effectiveness would suffer.

The process according to the invention, the reagent and the reagent kitenable for the first time the specific inactivation of serine proteasesin protein mixtures without changing the other proteins that are presentin this process. This is above all of particular interest with regard tothe production of proteins by genetic engineering for diagnostic andtherapeutic applications. Especially within the framework of theapproval of therapeutic proteins it is of major importance whether theproteins can be obtained in a pure form. If proteins are unspecificallyderivatized during the production process this can result in thecorresponding pharmaceutical preparation no longer meeting the approvalconditions and thus necessitating a new approval process. However, inthe process according to the invention for the inhibition of serineproteases, an unspecific derivatization is substantially and in somecases even completely avoided.

The present invention is further elucidated by the examples inconjunction with the figures.

FIG. 1 shows a measurement of the activity of chymotrypsin and theinhibition of chymotrypsin when various concentrations of Pefabloc(AEBSF) are added;

FIG. 2 shows the chymotrypsin inhibition with Pefabloc (AEBSF) in thepresence of hydroxypyridine; and

FIGS. 3 and 4 shows MALDI-TOF spectra which were recorded of insulintreated with AEBSF with (FIG. 3) or without (FIG. 4) addition of3-hydroxypyridine. FIG. 3 clearly shows the 4- to 5-fold unspecificderivatization of insulin on tyrosine and lysine residues with AEBSFwhereas only the insulin peak and a very weak peak for singlederivatization is seen in FIG. 4.

EXAMPLE 1

Monitoring the inhibition of chymotrypsin with AEBSF in the presence orabsence of 3-hydroxypyridine

Solution of chymotrypsin in 0.113 M Tris, 0.02 M CaCl2, pH 8.0 with orwithout 3-hydroxypyridine. In quick successionSuc-Ala-Ala-Pro-Phe-4-nitro-anilide was added as the substrate (0.19 mMin the test) and an AEBSF solution in 0.113M Tris, 0.02 M CaCl₂, pH 8.0was added to a final concentration in the test of 4-0.5 mM. Subsequentlythe reaction is immediately monitored by the change of absorbance at 405nm versus time. FIG. 1 and FIG. 2 show that the kinetics of theinhibition are not changed.

EXAMPLE 2

Reaction of insulin with AEBSF with or without 3-hydroxypyridine andmonitoring of the non-specific reactions by MALDI-TOF analysis.

0.1 ml aqueous insulin solution (1 mg/ml) is added to 2.5 ml of afreshly prepared solution of 0.113 M Tris, 0.02 M CaCl₂, 4 mM AEBSF, 15mM 3-hydroxypyridine, pH 8.0. The reaction is monitored by MALDI-TOF.For this 5 μl of the reaction mixture is removed each time and mixedwith 5 μl of a matrix solution (50 mM sinapic acid in 70 % acetonitrile,0.1 % TFA) of which 2 μl is applied to the target for examination.Additional signals soon appear in the absence of 3-hydroxypyridine. Inattachment 2 the reaction is shown after 21 hours reaction time.However, these secondary reactions are substantially inhibited with3-hydroxypyridine (see FIG. 3 and 4).

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 1 <210> SEQ ID NO: 1 <211>LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(4) <223>OTHER INFORMATION: chymotrypsin specific-substrate <221> NAME/KEY:misc_feature <222> LOCATION: (1)..(1) <223> OTHER INFORMATION: Bound tosuccinyl <221> NAME/KEY: misc_feature <222> LOCATION: (4)..(4) <223>OTHER INFORMATION: Bound to nitroanalide <400> SEQUENCE: 1 Ala Ala ProPhe

What is claimed is:
 1. A process for inhibiting serine proteases in asample comprising the steps: (i) adding to said sample a compound of thegeneral formula (I) and

in which X denotes C or N, R₁ denotes H, methyl, ethyl or OH, R₂ and R₃each denote H, methyl, ethyl, OH or residues which together form a ringclosure with a maximum of 6 C atoms in the ring and R₄ denotes H, OH oran optionally substituted alkyl, alkenyl or alkinyl group with up to 4 Catoms provided that at least one of R₁, R₂, R₃ or R₄ represents an OHgroup and the compound has a pKa of at least 8 and (ii) adding to saidsample a compound selected from the group consisting of sulfonic acidderivatives and fluorophosphonates, to derivative said serine proteases,wherein said compound (I) avoids nonspecific derivatization of proteinsother than said serine proteases.
 2. The process as claimed in claim 1,wherein a cresol or 3-hydroxypyridine is used as the compound of thegeneral formula (I).
 3. The process as claimed in claim 2, whereinp-cresol is used as the cresol.
 4. The reagent kit for inhibiting serineproteases containing in separate vessels a) a sulfonic acid derivativeor a fluorophosphate and b) a compound of the general formula (I)

in which x denotes C or N, R₁ denotes H, methyl, ethyl or OH, R₂ and R₃each denote H, methyl, ethyl, OH or residues which together form a ringclosure with a maximum of 6 C atoms in the ring and R₄ denotes H, OH oran optionally substituted alkyl, alkenyl or alkinyl group with up to 4 Catoms provided that at least one of R₁, R₂, R₃ or R₄ represents an OHgroup and the compound has a pKa of at least
 8. 5. The reagent kit asclaimed in claim 4, wherein the sulfonic acid derivative orfluorophosphate is 4-(2-aminoethyl)-phenyl-sulfonyl fluoridehydrochloride, diisopropyl fluorophosphate (DFP),amidinophenylmethylsulfonyl fluoride (APMSF) or phenylmethylsulfonylfluoride and the compound of the general formula (I) is cresol or3-hydroxypyridine.
 6. The reagent kit as claimed in claim 5, wherein thecresol is p-cresol.